PB1857: NRF2 MODULATORS IN CHRONIC LYMPHOCYTIC LEUKEMIA PATIENTS: A POSSIBLE NEW THERAPEUTIC APPROACH
Authors: J. P. Carda #, R. Gomes #, J. Jorge, A. C. Gonçalves, B. Marques, R. Alves, A. B. Sarmento-Ribeiro
Journal: HemaSphere (2022)

Background: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment needs. Oxidative stress (OS) is one of the mechanisms important in CLL development and progression. OS state consists in an unbalance between the production of reactive oxygen species (ROS) and the ability to neutralize them by antioxidant defenses. The nuclear factor erythroid 2–related factor 2, NRF2, is a transcription factor that regulates the expression of several antioxidants and detoxification systems and works as a protective mechanism in normal cells against malignant transformation. However, during the tumorigenic process, NRF2 protects tumor cells from OS effects compromising the therapeutic efficacy. Aims: The study aims to evaluate the therapeutic potential of two NRF2 modulators, Brusatol and ML385, in B cells from CLL patients. Methods: Peripheral blood mononuclear cells (PBMCs) from 13 CLL patients (stage initial, Binet A/B, n=8; advanced stage, Binet C, n=5) were isolated by density gradient using Ficoll-Paque Premium. PBMCs where immediately incubated in absence and presence of Brusatol and ML385. The cytotoxic effect was evaluated 24h and 48h after incubation by fluorometric microculture cytotoxicity assay. After 24h incubation with Brusatol (100nM) and ML385 (100μM) cells were stained with anti-CD5-PerCP, anti-CD19-APC and Annexin V, to assess cell death by flow cytometry. The statistical analysis was carried out in the total CLL patient group and in risk groups. The results were considered statistically significant when p<0.05. Results: CLL patients presented 93,3% (±3.7%) of neoplastic B cells (CD5+/CD19+), 3,7% (±3,0%) of normal B cells (CD5-/CD19+) and 2,7% (±1,2%) of normal T cells (CD5+/CD19-), with no statistical differences regarding staging groups. Brusatol induced a decreased survival rate in CLL cells comparing to ML385, being this effect dependent on the dose and disease stage. Brusatol induced a higher reduction of cell viability in PBMCs from advanced disease patients (Binet C). ML385 did not induced a significant decrease in cell survival, nevertheless its effect is more marked in advanced stage patients at 48h post incubation. IC50 was only achieved at 48h in advanced stage patients and corresponds to 65nM for Brusatol and to 114µM for ML385. NRF2 modulators induced a cytotoxic effect mainly mediated by apoptosis. Cytotoxicity induced by both drugs was more evident in neoplastic B cells, with no significant cytotoxicity in normal B and T cells. In PBMCs incubated with Brusatol an increase of neoplastic B cells in apoptosis about 68% was observed (Brusatol: 91,9% ± 7,1%; Control: 23,7% ± 4,3%), and of 34% in cells incubated with ML385 (ML385: 57,9% ± 10,2%; Control: 23,7% ± 4,3%). No differences of apoptotic cells were observed in advanced stage groups. Summary/Conclusion: Our results showed that neoplastic cells from CLL patients were sensitive to NRF2 modulators, suggesting that these drugs may constitute a new therapeutic approach in CLL patients, particularly in CLL patients in advanced stage. We also demonstrated the low cytotoxic effect of these modulators in normal lymphocytes and its higher efficacy in neoplastic cells from advanced stage patients. Altogether, these results show that these NRF2 modulators have an effect directed to the neoplastic cell and suggest a possible involvement of NRF2 on CLL development and progression. # equally contributing authors.